Late stage ovarian cancer is marked by poor patient survival and significant metastatic spread throughout the peritoneal cavity. During transcoelomic spread cells must adapt to survive anchorage-independence and to cope with stress associated with matrix detachment and the hostile environment of the ascites. Preliminary findings demonstrate that an important ovarian cancer cell adaptation during anchorage-independence is the up- regulation of two mitochondrial proteins, superoxide dismutase 2 (Sod2), and its regulator, the metabolic and redox sensing deacetylase SIRT3. Moreover, these proteins are necessary for anchorage-independent cell survival and transcoelomic metastasis in vivo. In addition to Sod2?s role as a mitochondrial superoxide scavenger, mechanistic data show that Sod2 shifts the redox landscape of cancer cells to a higher hydrogen peroxide (H2O2) steady-state. This novel non-canonical function of Sod2 as a H2O2 regulator results in mitochondrial redox signaling, as demonstrated by oxidation and inactivation of phosphatases, and enhanced Akt signaling. Preliminary data demonstrate that SIRT3 is an important regulator of Sod2 during anchorage- independence, which points to a novel pro-survival role for SIRT3 during ovarian cancer spread. Thus, the proposal will test the hypothesize that mitochondrial redox signaling is an important regulator of survival adaptations in response to matrix detachment, and that two key mitochondrial proteins, SIRT3 and Sod2, are required for the initiation and regulation of mitochondrial redox signaling in anchorage-independence. This will be addressed using a combination of cell culture models, patient ascites-derived cells, in vivo mouse xenograft models, and molecular, biochemical and imaging techniques to monitor oxidants and redox signaling. Using both unbiased screens and targeted approaches Aim 1 consists of mechanistic studies to delineate how SIRT3/Sod2- regulated mitochondrial H2O2-signaling drives anchorage-independent survival. In Aim 2 the mechanisms of SIRT3 activity and transcriptional regulation will be elucidated how SIRT3 acts as the sensor of matrix detachment in the context of metabolic changes associated with anchorage-independence. In proof-of-principle studies of Aim 3 the vulnerability of Sod2-high tumor cells will be targeted with two approaches based exploiting their sensitivity to H2O2 generating agents and Akt inhibition. Establishing that mitochondrial redox signaling is a necessary adaptation for ovarian cancer anchorage-independent survival and metastasis is crucial in our long- term goal of targeting key metastatic adaptations for novel therapies against ovarian cancer.